Rapid Delivery of Nanobodies/V(H)Hs into Living Cells via Expressing In Vitro-Transcribed mRNA

Intracellular antigen labeling and manipulation by antibodies have been long-thought goals in the field of cell research and therapy. However, a central limitation for this application is that antibodies are not able to penetrate into the cytosol of living cells. Taking advantages of small sizes and unique structures of the single-domain antibodies, here, we presented a novel approach to rapidly deliver the nanobody/variable domain of heavy chain of heavy-chain antibody (V(H)H) into living cells via introducing its coding mRNA, which was generated by in vitro transcription. We demonstrated that actin-green fluorescent proteins (GFP) and Golgi-GFP can be recognized by the anti-GFP nanobody/V(H)H, vimentin can be recognized by the anti-vimentin nanobody/V(H)H, and histone deacetylase 6 (HDAC6) can be recognized by the anti-HDAC6 nanobody/V(H)H, respectively. We found that the anti-GFP nanobody expressed via in vitro-transcribed (IVT) mRNA can be detected in 3 h and degraded in 48 h after transfection, whereas the nanobody expressed via plasmid DNA, was not detected until 24 h after transfection. As a result, it is effective in delivering the nanobody through expressing the nanobody/V(H)H in living cells from its coding mRNA.